Tumor cells communicate with their surroundings via interactions between their transmembrane proteins (TmPs) and host extracellular factors such as soluble ligands or matrix proteins: communications that have long been linked to cancer progression and metastasis. To identify TmPs that foster aggressive behavior of tumor cells we selected several pairs of congenic human tumor cell variants differing in their capacity of spontaneous metastasis and by using these variants in subtractive approaches, identified a number of TmPs that have been strongly linked to metastasis. Importantly, the function of one of these TmPs, CDCP1, has been linked to its ectodomain cleavage on the tumor cell surface, leading to a novel hypothesis that specific proteolytic modifications of certain TmPs determine survival of escaping primary tumor cells. To investigate this hypothesis and test its diagnostic and translational potential, we propose in Aim 1 to: Elucidate a targetable mechanism to control tumor cell survival and metastatic colonization enhanced by plasmin-mediated cleavage of the transmembrane protein, CDCP1. We have demonstrated that the 70-kDa membrane-retained CDCP1 fragment becomes phosphorylated and induces survival signaling allowing escaping primary tumor cells to avoid apoptosis and successfully complete colonization. We also have developed a mouse lung retention assay that uniquely allows for simultaneous quantification of in vivo cleaved CDCP1, de novo generated plasmin, survival of CDCP1-expressing metastatic cells, and proteolysis-induced signaling cascades in lung-retained tumor cells. Therefore we will: (1) identify the molecules that regulate in vivo plasminogen activation and coordinated plasmin-mediated cleavage of tumor cell CDCP1; (2) determine the tissue locale and initiating events for plasmin generation linked specifically to CDCP1 cleavage; (3) elucidate how cell surface cleavage of CDCP1 initiates in vivo signal transduction enhancing tumor cell survival; and (4) examine the translational potential of detecting CDCP1 and its cleaved fragments in the histopathology of human prostate cancer. We propose in Aim 2 to: Establish a general mechanism whereby limited cleavage of distinct cell surface TmPs by targetable host proteases determines the in vivo survival of tissue-retained tumor cells. By employing genetically-defined mice lacking the expression of specific host proteases, we will: (1) directly link the in vivo survival of tissue-retained tumor cells to their metastatic capability; (2) identify specific host proteases that increase survival rates of highly malignant tumor cells; (3) identify new TmPs, cell surface cleavage of which enhances tumor cell survival; and (4) investigate the in vivo functional role of MMP-9-mediated CD44 cleavage in tumor cell vascular survival and metastatic colonization. The proposed research will ultimately prove that irreversible modifications of distinct TmPs determine their in vivo functionality, thereby affecting overall metastatic outcome and representing an important translational aspect ultimately aimed for development of diagnostic or and/or risk stratification tools aiding cancer patient therapy.